Francisella tularensis is a gram-negative bacterium and the causative agent of tularemia, a potentially deadly disease. It is a threat as an agent of bioterrorism because of the highly infectious nature of the airborne organism, with as few as 10 organisms being sufficient to cause disease. In the mammalian host, the bacterium is an intracellular pathogen, replicating within membrane-bound phagosomes in macrophages and other cells. Iron is a scarce but essential nutrient for the invading pathogen, and iron acquisition systems are proving to be critical virulence determinants. The availability of iron was shown to be important for proliferation of F. tularensis within macrophages by Fortier et al. (Inf. Immun.63:1478, 1995). Iron acquisition mechanisms in this organism are however uncharacterized. The goal of this proposal is identification of systems for iron acquisition in F. tularensis with the hypothesis that iron acquisition is a key element in pathogenesis in the mammalian host. A variety of techniques including bioinformatics (based on the genome sequencing project currently underway), proteomic and genetic approaches will be taken to achieve this goal. Specific aim 1 of this project is the characterization of iron utilization in the organism. By analyzing growth of the bacteria under conditions of iron-limitation, characterizing uptake of ferrous versus ferric iron, and determining the various host sources of iron (transferrin, heme etc.) that can be utilized, the physiologically significant sources of iron for the invading pathogen may be identified. These studies will help define assay methods for studying specific transport mechanisms. Specific aim 2 is the identification of iron acquisition systems and characterizing their role in virulence. Identification of potential transport systems will involve bioinformatics-based and proteomic approaches followed by targeted mutagenesis. Mutants will be tested for their ability to proliferate in a macrophage infection assay as a first level determination of their role in virulence. The long-term goals with these studies are to explore potential new mechanisms for iron acquisition, to be able to harness them in therapeutic applications, and to test them as targets for a defined vaccine strain development.